The overall goal of this project is to investigate strategies for the drug treatment of epilepsy through pharmacological studies in animal models and clinical investigation in human subjects. Research was continued evaluating the role of neuroactive steroids in epilepsy and their possible uses in epilepsy therapy. Neuroactive steroids are endogenous steroid hormones (and their synthetic analogs) that rapidly alter the excitability of neurons by direct actions on membrane ion channels, including GABA-A and NMDA receptors. In prior reporting periods, we confirmed that the reproductive hormone progesterone has powerful anticonvulsant activity. Studies with progesterone receptor knockout (PRKO) mice demonstrated that the effects of progesterone on seizure susceptibility are not mediated by its cognate nuclear hormone receptors. Instead, we found that progesterone is anticonvulsant as a result of its conversion to the neuroactive steroid allopregnanolone. We proposed that perimenstrual catamenial epilepsy, the increase in seizure frequency that many women with epilepsy experience near the time of menstruation (when progesterone levels fall) may be related to withdrawal of allopregnanolone. At present, there is no specific treatment for catamenial epilepsy. However, our studies with an animal model of catamenial epilepsy suggested that neurosteroid replacement could be useful. In addition, we have investigated the role of neurosteroids in stress-induced alterations in seizure susceptibility, focusing specifically on deoxycorticosterone (DOC), an adrenal steroid whose synthesis is enhanced during stress. Our results demonstrated that DOC is a mediator of the physiological effects of acute stress that could contribute to stress-induced changes in seizure susceptibility through its conversion to neurosteroids with modulatory actions on GABA-A receptors including tetrahydrodeoxycorticosterone (THDOC). Our results further suggest a role for neuroactive steroids as a treatment approach for stress-related seizures. In the present reporting period, we conducted studies using progesterone receptor knockout (PRKO) mice to evaluate the role of the progesterone receptor (PR) in the anxiolytic activity of progesterone. These studies are complementary to research reported previously in which we used PRKO mice to examine the role of PR in the anticonvulsant activity of progesterone. The absence of PR receptor protein expression in PRKO brain was confirmed by immunocytochemistry. In PRKO mice and their isogenic wild-type (WT) littermates, progesterone administration was associated with a dose-dependent rise in plasma allopregnanolone concentrations and corresponding anxiolytic effects in the elevated plus maze test. PRKO mice exhibited a greater anxiolytic response than WT animals although the allopregnanolone levels were similar in the two genotypes. Allopregnanolone also exhibited anxiolytic effects, but the magnitude of the response was similar in both genotypes. Pretreatment of PRKO mice with finasteride, a 5alpha-reductase inhibitor that blocks the conversion of progesterone to allopregnanolone, completely prevented the anxiolytic activity of progesterone, but had no effect on the response to allopregnanolone, demonstrating that allopregnanolone (or other 5alpha-reduced metabolites of progesterone) accounts for the response to the parent steroid hormone. Our results provide direct evidence that the anxiolytic action of progesterone does not require PRs. However, PR activation by progesterone may influence the anxiolytic response since PRKO mice were more sensitive to progesterone. Neuroactive steroids may be preferred to progesterone in the treatment of catamenial epilepsy because neuroactive steroids do not have hormonal actions mediated by PRs which can lead to side effects. The present results provide further support for the use of neuroactive steroids rather than progesterone in the treatment of catamenial epilepsy, since progesterone can act on brain PR receptors and potentially produce undesirable effects on brain function. During this reporting period we also began studies examining the role of androgen-related steroids in the regulation of seizure susceptibility. Men with epilepsy often have sexual or reproductive abnormalities that are attributed to alterations in androgen levels, including subnormal free testosterone. Levels of the major metabolites of testosterone--androsterone (5alpha-androstan-3alpha-ol-17-one), a neurosteroid that acts as a positive allosteric modulator of GABA-A receptors, and its 5beta-epimer etiocholanolone (5beta-androstan-3alpha-ol-17-one)--also may be reduced in epilepsy. Androsterone has been found in adult brain, and both metabolites, which also can be derived from androstenedione, are present in substantial quantities in serum along with their glucuronide and sulfate conjugates. We hypothesized that androsterone and etiocholanolone have anticonvulsant properties, so that low levels of these metabolites in men with epilepsy could lead to enhanced seizure susceptibility and poor seizure control. Supporting evidence was obtained in animal epilepsy models and in an in vitro brain slice model of epileptiform activity. Androsterone protected mice in a dose-dependent fashion from seizures in the 6-Hz electrical stimulation, pentylenetetrazol, pilocarpine, 4-AP, and maximal electroshock models. Etiocholanolone also had anticonvulsant properties, but was less potent, whereas epiandrosterone (3beta-androsterone) was inactive. Androsterone also inhibited epileptiform discharges in hippocampal slices exposed to 4-aminopyridine, whereas etiocholanolone was active but of lower potency. We conclude that androsterone and etiocholanolone have anticonvulsant properties and could represent endogenous modulators of seizure susceptibility. Reductions in the levels of these metabolites in men with epilepsy could contribute to poor seizure control. As in catamenial epilepsy, neuroactive steroid replacement is a potential therapeutic strategy.